comparativeanalysisofviperidaevenomsantibacterialproﬁle...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2011, Article ID 960267, 4 pagesdoi:10.1093/ecam/nen052

Original Article

Comparative Analysis of Viperidae Venoms Antibacterial Profile:a Short Communication for Proteomics

Bruno L. Ferreira,1, 2 Dilvani O. Santos,1, 2 Andre Luis dos Santos,1, 2 Carlos R. Rodrigues,2, 3

Cıcero C. de Freitas,1 Lucio M. Cabral,3 and Helena C. Castro1, 2

1 Departamento de Biologia Celular e Molecular, Laboratorio de Antibioticos, Bioquımica e Modelagem Molecular (LABioMol),Instituto de Biologia, CEG, Universidade Federal Fluminense, CEP 24001–970, Niteroi, Brazil

2 Cursos de Pos-graduacao em Neuroimunologia - IB, e Patologia - HUAP, Universidade Federal Fluminense, CEP 24001-970,Niteroi, RJ, Brazil

3 Laboratorio de Modelagem Molecular e QSAR (ModMolQSAR), Faculdade de Farmacia, Universidade Federal do Rio de Janeiro,CEP 21941-590, Rio de Janeiro, RJ, Brazil

Correspondence should be addressed to Helena C. Castro, [email protected]

Received 2 May 2007; Accepted 16 July 2008

Copyright © 2011 Bruno L. Ferreira et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Bacterial infections involving multidrug-resistant strains are one of the ten leading causes of death and an important healthproblem in need for new antibacterial sources and agents. Herein, we tested and compared four snake venoms (Agkistrodonrhodostoma, Bothrops jararaca, B. atrox and Lachesis muta) against 10 Gram-positive and Gram-negative drug-resistant clinicalbacteria strains to identify them as new sources of potential antibacterial molecules. Our data revealed that, as efficient as someantibiotics currently on the market (minimal inhibitory concentration (MIC) = 1–32 µg mL−1), A. rhodostoma and B. atrox venomswere active against Staphylococcus epidermidis and Enterococcus faecalis (MIC = 4.5 µg mL−1), while B. jararaca inhibited S. aureusgrowth (MIC = 13 µg ml−1). As genomic and proteomic technologies are improving and developing rapidly, our results suggestedthat A. rhodostoma, B. atrox and B. jararaca venoms and glands are feasible sources for searching antimicrobial prototypes forfuture design new antibiotics against drug-resistant clinical bacteria. They also point to an additional perspective to fully identifythe pharmacological potential of these venoms by using different techniques.

1. Introduction

Bacterial infections are among the 10 leading causes of deathworldwide according to the World Health Organization [1,2]. The presence and current emergence of the kept multiple-resistant strains make the risk of these infections becomemore threatening as the treatment becomes unreachable. Infact, bacterial resistance has been the major factor responsi-ble for increasing morbidity, mortality and health care costsof bacterial infections [1–5]. Therefore, new antimicrobialsor antibacterial prototypes are continuously necessary fordrug design and development for treatment of infectionsinvolving multidrug-resistant microorganisms [1, 2, 6, 7].

Snake venoms are a complex mixture of proteins andpeptides that display potential biological activities and maylead to the production of new drugs of potential therapeutic

value [8, 9]. A good example is the bradykinin-potentiatingpeptides (BPPs), which are naturally occurring inhibitors ofthe somatic angiotensin-converting enzyme (ACE) found inBothrops jararaca venom [10]. The chemical and pharma-cological properties of these peptides were essential for thedevelopment of captopril, the first active site directedinhibitor of ACE, currently used to treat human hyperten-sion [11].

Lately, several naturally occurring peptides presentingantimicrobial activity have been described in the literature.However, Viperidae snake venoms that are an enormoussource of peptides have not been fully explored for searchingsuch biological activity [5, 12]. Therefore, in this study,we tested the antibiotic profile of four snake venomsfrom three different genera of Viperidae family (Agkistrodonrhodostoma, Bothrops atrox, B. jararaca and Lachesis muta)

2 Evidence-Based Complementary and Alternative Medicine

against 10 drug-resistant Gram-positive and Gram-negativebacteria clinical strains. Based on captopril history, ourpurpose is to identify some of these venoms as potentialsource for finding new and effective antibacterial prototypes.

2. Materials and Methods

2.1. Materials. Each venom was purchased from Sigma (StLouis, MO, USA) and one L. muta venom sample was alsoobtained from each Brazilian governmental sources—Butantan Institute, SP, Ezequiel Dias Institute, SP and VitalBrazil Institute, RJ, Brazil. The 10 Gram-positive (Ente-rococcus faecalis, Staphylococcus epidermidis and S. aureus)and Gram-negative (Escherichia coli, Serratia marcenses, Pro-teus mirabilis, Pseudomonas aeruginosa, Enterobacter cloacae,Acinetobacter calcoaceticus and Klebsiella pneumoniae) drug-resistant clinical bacteria were isolated from patients of theHospital Antonio Pedro from Fluminense Federal University.All other reagents were from Sigma. After isolation, thebacterial strains were kept frozen in 10% milk-sterilizedsolution containing 10% glycerin.

2.2. Methods

2.2.1. Sensibility Test. The test and MIC were performedaccording to the National Committee for Clinical Labo-ratory Standards (NCCLS), in Mueller-Hinton medium asdescribed elsewhere [13]. Briefly, the strains were grownat 37◦C in Mueller-Hinton medium. Then, 1 µL of thesnake venom solutions prepared with sterilized distilled anddeionized water (20 mg mL−1) was placed in Whatman disks(5 mm diameter). The disks were put on an exponentiallygrowing plated culture with appropriate dilution to 1.0 ×107 colony forming unit (CFU mL−1), which were thenincubated for 24 h at 37◦C. The inoculums used in growthmethod were those where turbidity was equal to 0.5 McFar-land Standard. The results were verified by measuring thezones surrounding the disk. Ciprofloxacin and vancomycinwere used as positive controls and the halo >15 mm wasconsidered the minimum value for positive antimicrobialactivity as it generally leads to a MIC near to that observedfor the newest antimicrobials current present on the market(MIC = 1−40 µg mL−1). Vancomycin and ciprofloxacinpresented halo 15 ± 2 and 23 ± 2 mm, respectively in thestrains tested herein (P < .005).

2.2.2. MIC Assays. In order to determine the MIC of thesesnake venoms, we tested them using the macro-dilutionbroth method as described elsewhere [13]. Briefly, after 5 h ofthe bacterial growth, the culture was diluted to obtain 1.0 ×105 CFU mL−1. The snake venom was added in order to reacha final concentration from 0.5 µg mL−1 to 1024 µg mL−1

and incubated at 37◦C for 24 h. MIC was defined as thelowest concentration of venom or antibiotic current onthe market preventing visible bacterial growth compared tothe positive growth control (medium plus bacteria withoutvenom or antibiotic) that presented high turbidity, andto the negative growth controls (medium alone, medium

Table 1: Antibacterial effect of Viperidae venoms against Gram-positive and Gram-negative drug-resistant clinical bacteria.

StrainInhibition zone (mm)a

A. rhodostoma B. atrox B. jararaca L. muta

E. faecalis 16± 2 16± 2 13 ± 1 1 ± 1

S. epidermidis 16± 1 18± 2 0 0

S. aureus 13 ± 2 12 ± 1 16± 1 2 ± 1

E. coli 11 ± 1 7 ± 1 5 ± 2 0

S. morcencens 8 ± 1 8 ± 2 7 ± 1 1 ± 1

P. mirabilis 7 ± 1 7 ± 1 7 ± 1 2 ± 1

P. aeruginosa 8 ± 2 8 ± 1 8 ± 1 1 ± 1

E. calcoacetic 9 ± 1 6 ± 2 6 ± 2 2 ± 1

A. calcoacetic 1 ± 2 2 ± 1 1 ± 1 1 ± 1

K. pneumoniae 9 ± 1 10 ± 1 10 ± 2 0aThe values represent a venom inhibition zone in millimeters, after l8 hincubation performed in triplicate assays (P < .005). Significant results wereconsidered the halo >15 mm as vancomycin and ciprofloxacin presentedhalo = 15–17 and 23–25 mm, respectively.

plus venom or antibiotic and medium plus bacteria pluseffective antibiotic) that presented no turbidity. All strainswere tested at least in duplicate in four separate experimentsand a reference antibiotic (vancomycin) was used as standard(MIC = 2 µg mL−1).

2.3. Statistics. The statistical analyses were performed usingMicrocal Origin 4.0 (MA, USA). The SD and significance(P) were determined by using one-way ANOVA from thesame graphic program. Results were considered as significantwhen P < .005.

3. Results

3.1. Sensibility Assays of Venoms. Our experimental datarevealed that most of the venoms tested (A. rhodostoma,B. atrox and B. jararaca) exhibited an antibacterial profileagainst some of the Gram-positive bacteria (Table 1). TheL. muta venom showed no antibacterial activity (Table 1)even when it was obtained from different Brazilian suppliers(Instituto Butantan, Ezequiel Dias and Vital Brazil) (data notshown).

Agkistrodon rhodostoma was not significantly effectiveagainst S. aureus and E. coli (Table 1). However, this venomwas able to significantly inhibit E. faecalis and S. epidermidisgrowth (halo = 16 and 16 mm, resp.). Bothrops atrox venomalso showed an antibiotic profile against E. faecalis and S.epidermidis (halo = 16 and 18 mm, resp.), different from B.jararaca venom, which acted only against S. aureus (halo =16 mm) (Table 1). In addition, Bothrops venoms (B. atroxand B. jararaca) inhibited growth of Staphylococcus sp. (S.epidermidis and S. aureus, resp.).

3.2. MIC Assays of the Active Snake Venoms. The MIC assaysof the snake venoms that were active in the sensibility tests(A. rhodostoma, B. atrox and B. jararaca) revealed that their

Evidence-Based Complementary and Alternative Medicine 3

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20

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Figure 1: MIC of Viperidae venoms presenting halo >15 mm.

Table 2: MIC of Viperidae venoms presenting halo >15 mm.

Straina MIC (µg mL−1)

A. rhodostoma B. atrox B. jararaca

E. faecalis 4.5 4.5 —

S. epidermidis (A) 4.5 4.5 —

S. epidermidis (B) 4.5 4.5 —

S. aureus (A) — — 13

S. aureus (B) — — 13a(A) and (B) on S. aureus and S. epidermidis refer to different patients

strains.

level of antibiotic activity was comparable (MIC = 4.5–13 µg mL−1) (Table 2). Agkistrodon rhodostoma and B. atroxwere also analogous to the antibiotics currently in use againstS. epidermidis, such as ampicillin, chloramphenicol, van-comycin, oxacillin and penicillin G (MIC = 1–32 µg mL−1)(Figure 1).

4. Discussion

Our experimental data revealed that most of the venomstested (A. rhodostoma, B. atrox and B. jararaca) exhibited apromising antibacterial activity against some of the Gram-positive bacteria. Interestingly, despite of the presence of aknown phospholipase A2 [14], the L. muta venom showedno antibacterial activity. Although literature described thatdifferent snake may present an individual pattern [5], L.muta different venom samples obtained from three differ-ent Brazilian suppliers (Governmental Institutes—Butantan,

Ezequiel Dias and Vital Brazil) were not able to affectthe bacterial strains (data not shown). This negative resultreinforce the fact that the presence of enzymes in the snakevenoms do not guarantee the antibiotic profile of thesematerials as the bacteria cell wall may avoid or affect theactions of these proteins against them.

Differently from A. contortrix venom [12], A. rhodostomavenom was able to significantly inhibit E. faecalis and S.epidermidis growth, which may suggest a specific mechanismor molecule of A. rhodostoma on affecting them.

Bothrops atrox venom also showed an antibiotic profileagainst E. faecalis and S. epidermidis, different from B.jararaca venom, which acted only against S. aureus. Recently,the literature described l-amino acid oxidases (L-MAO)isolated from B. pirajai [15] and B. alternatus venoms[16] able to inhibit E. coli growth. Our result pointed tothe L-MAO isoforms presence in Bothrops sp. venoms aspreserved components similar to C-type lectin-like proteins[17]. However, as the active profile of these venoms switchedto different strains and not included E. coli, our result mayalso suggest that other antibacterial components may bepresent in these venoms resulted from species differentiation.In addition, Bothrops venoms (B. atrox and B. jararaca)acted against different Staphylococcus sp. (S. epidermidis andS. aureus, respectively) once again suggesting that differentmolecules and/or targets are involved in these biologicalactivities. This hypothesis is reinforced by the presence ofother different components found in snake venoms thatsometimes are involved in a similar biological activity asRGD-peptides and some C-type lectin-like proteins from B.jararaca venom that are both platelet aggregation inhibitors[9].

Overall, our MIC assays revealed that the activity ofthe venoms tested was comparable (MIC = 4.5–13 µg mL−1)among them. Agkistrodon rhodostoma and B. atrox were alsoanalogous to the antibiotics currently in use against S. epi-dermidis, such as ampicillin, chloramphenicol, vancomycin,oxacillin and penicillin G (MIC = 1−32 µg mL−1). Presently,S. epidermidis is an important nosocomial pathogen, dras-tically affecting immunocompromised patients and/or thosewith indwelling devices, such as joint prostheses, prostheticheart valves and central venous catheters [18]. Therefore,these venoms’ active profile against this strain is of interestfor pursuing continuously an antibacterial molecule.

Proteomic technologies are improving and developingrapidly [19]. An important goal of proteomic studies ofsnake venoms is discovering molecules that may be used intreatment of diseases or as drugs prototypes. Nevertheless,these techniques may depend on the experimental datagenerated so far to identify some of these unknown proteinsor peptides. Although snake venom peptides and proteinshave a limited direct therapeutical use due to their antigenicand “digestible” structure, their usefulness as prototypeshas clear potential [9, 20]. Our data suggested that A.rhodostoma, B. atrox and B. jararaca are feasible sourcesfor searching antimicrobial prototypes and designing newantibiotics against drug-resistant clinical bacteria, and thesedata may act as a start for investing on these venomsproteomic study for prototypes searching.

4 Evidence-Based Complementary and Alternative Medicine

Acknowledgments

The authors would like to thank Conselho Nacional de Des-envolvimento Cientıfico e Tecnologico (CNPq), Universi-dade Federal Fluminense (UFF) and Fundacao de Amparoa Pesquisa do Estado do Rio de Janeiro (FAPERJ) from Brazilfor the financial support and fellowships of H.C.C. They alsothank L. C. Correa for his technical assistance.

References

[1] A. D. Lopez, C. D. Mathers, M. Ezzati, D. T. Jamison, andC. J. L. Murray, in Global Burden of Disease and Risk Factors,IBRD/The World Bank and Oxford University Press, Washing-ton, DC, USA, 2006.

[2] World Health Organization, WHO, Department of Com-municable Disease Surveillance, World Health Organization,Department of Essential Drugs and Medicines Policy, January2007, http://www.who.int/en/.

[3] T. M. Barbosa and S. B. Levy, “The impact of antibiotic useon resistance development and persistence,” Drug ResistanceUpdates, vol. 3, no. 5, pp. 303–311, 2000.

[4] J. Y. Ang, E. Ezike, and B. I. Asmar, “Antibacterial Resistance,”Indian Journal of Pediatrics, vol. 71, no. 3, pp. 229–239, 2004.

[5] D. C. de Lima, P. Alvarez Abreu, C. C. de Freitas, D. O. Santos,R. O. Borges, T. C. Dos Santos et al., “Snake venom: any cluefor antibiotics and CAM?” Evidence-Based Complementaryand Alternative Medicine, vol. 2, pp. 39–47, 2005.

[6] L. Guardabassi and H. Kruse, “Overlooked aspects concerningdevelopment and spread of antimicrobial resistance,” ExpertReview of Anti-Infective Therapy, vol. 1, no. 3, pp. 359–362,2003.

[7] K. L. Roos, “Emerging antimicrobial-resistant infections,”Archives of Neurology, vol. 61, pp. 1512–1514, 2004.

[8] J. White, “Bites and stings from venomous animals: a globaloverview,” Therapeutic Drug Monitoring, vol. 22, no. 1, pp. 65–68, 2000.

[9] D. C. Koh, A. Armugam, and K. Jeyaseelan, “Snake venomcomponents and their applications in biomedicine,” Cellularand Molecular Life Sciences, vol. 63, pp. 3030–3041, 2006.

[10] S. H. Ferreira, D. C. Bartelt, and L. J. Greene, “Isolationof bradykinin-potentiating peptides from bothrops jararacavenom,” Biochemistry, vol. 9, no. 13, pp. 2583–2593, 1970.

[11] G. L. Plosker and D. McTavish, “Captopril. A review ofits pharmacology and therapeutic efficacy after myocardialinfarction and in ischaemic heart disease,” Drugs & Aging, vol.7, pp. 226–253, 1995.

[12] B. G. Stiles, F. W. Sexton, and S. A. Weinstein, “Antibacterialeffects of different snake venoms: purification and charac-terization of antibacterial proteins from Pseudechis australis(Australian king brown or mulga snake) venom,” Toxicon, vol.29, pp. 1129–1141, 1991.

[13] C. G. T. Oliveira, F. F. Miranda, V. F. Ferreira et al., “Synthesisand antimicrobial evaluation of 3-hydrazino-naphthoquino-nes as analogs of lapachol,” Journal of the Brazilian ChemicalSociety, vol. 12, no. 3, pp. 339–345, 2001.

[14] A. L. Fuly, A. L. P. De Miranda, R. B. Zingali, and J. A.Guimaraes, “Purification and characterization of a phospholi-pase A2 isoenzyme isolated from Lachesis muta snake venom,”Biochemical Pharmacology, vol. 63, no. 9, pp. 1589–1597, 2002.

[15] L. F. M. Izidoro, M. C. Ribeiro, G. R. L. Souza et al., “Biochemi-cal and functional characterization of an l-amino acid oxidase

isolated from Bothrops pirajai snake venom,” Bioorganic andMedicinal Chemistry, vol. 14, no. 20, pp. 7034–7043, 2006.

[16] R. G. Stabeli, S. Marcussi, G. B. Carlos et al., “Platelet aggre-gation and antibacterial effects of an l-amino acid oxidasepurified from Bothrops alternatus snake venom,” Bioorganicand Medicinal Chemistry, vol. 12, no. 11, pp. 2881–2886, 2004.

[17] H. C. Castro, M. Fernandes, and R. B. Zingali, “Identificationof bothrojaracin-like proteins in snake venoms from Bothropsspecies and Lachesis muta,” Toxicon, vol. 37, no. 10, pp. 1403–1416, 1999.

[18] S. R. Gill, D. E. Fouts, G. L. Archer et al., “Insights on evolutionof virulence and resistance from the complete genome analysisof an early methicillin-resistant Staphylococcus aureus strainand a biofilm-producing methicillin-resistant Staphylococcusepidermidis strain,” Journal of Bacteriology, vol. 187, no. 7, pp.2426–2438, 2005.

[19] M. M. Elrick, J. L. Walgren, M. D. Mitchell, and D. C. Thomp-son, “Proteomics: recent applications and new technologies,”Basic and Clinical Pharmacology and Toxicology, vol. 98, no. 5,pp. 432–441, 2006.

[20] D. W. Cushman and M. A. Ondetti, “History of the designof captopril and related inhibitors of angiotensin convertingenzyme,” Hypertension, vol. 17, no. 4, pp. 589–592, 1991.

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